Seasonal Changes in Undifilum Colonizationand Swainsonine Content of Locoweeds

Jorge Achata Böttger & Rebecca Creamer &

Dale Gardner

Received: 5 December 2011 /Revised: 5 March 2012 /Accepted: 31 March 2012 /Published online: 28 April 2012# Springer Science+Business Media, LLC 2012

Abstract Locoweeds (Astragalus and Oxytropis) are legumi-nous plants that are toxic due to a symbiotic association with theendophytic fungus Undifilum oxytropis. The fungus producesthe alkaloid swainsonine, an α-mannosidase-inhibitor thatcauses serious damage to mammals when consumed. A real-time PCR technique was developed to quantify the colonizationextent of Undifilum in locoweeds and to compare it to theswainsonine concentration in the plants. Amplification of theendophyte nuclear ITS region allowed reliable quantification ofUndifilum DNA from field plants and in vitro cultures. Swain-sonine concentration was highly correlated (ρ00.972, P<0.001) with the proportion of Undifilum DNA during the first4 weeks of in vitro culture growth. Species of Astragalus andOxytropis were sampled seasonally in New Mexico and Colo-rado for two years. High swainsonine concentration in plantsamples was associated with high levels of endophyte DNA,except in plant reproductive tissues.

Keywords Undifilum . Quantitative PCR . Swainsonine

Introduction

Locoweeds are toxic legumes in the genera Astragalus andOxytropis (Marsh, 1909; Patterson, 1982; James and Panter

1989). They are toxic because of a mutualistic association withthe endophytic fungus Undifilum oxytropis (Braun et al., 2003;Ralphs et al., 2008; Pryor et al., 2009). The fungus producesswainsonine, an indolizidine alkaloid that inhibits α-mannosidases causing serious damage to mammals when con-sumed. Animals often develop behavioral changes after repeat-ed consumption of locoweed, as they becomemore intoxicated.Different types of physiological damage result from swainso-nine consumption, including changes in serum enzyme levelsand intracellular vacuolation in various tissues, which results inneurological disorders and reproductive complications (Ralphset al., 1993; McLain-Romero et al., 2004; Stegelmeier et al.,2005).

Astragalus species tend to be annuals depending on precip-itation, whereas Oxytropis species tend to have a perennial lifecycle (Ralphs et al., 2003). Locoism outbreaks of major eco-nomic importance caused by A. lentiginosus, A. emoryanus, A.wootonii, A. whawhaepensis, and A. pubentissimus have beenrecorded in North America (Williams et al., 1979; Molyneauxand James, 1982; Davis et al., 1984; Pfister et al., 2003; Ralphset al., 2003), some of which occur after heavy rainfall years.Locoweed seeds can remain viable for decades and currentlyare the only known dispersal mechanism for both the plant andthe endophyte (Ralphs et al., 2003). Undifilum colonizes theseed coat while in the ovary (Oldrup et al., 2010), and embryocolonization occurs later on, during germination. AlthoughU. oxytropis does not appear to induce disease to North Amer-ican locoweeds, recent reports describe a related fungus,Embellisia astragali, causing disease in A. adsurgens and A.sinicus in China (Li and Nan, 2007a, 2007b).

Rangeland management and exposure avoidance havebeen crucial in decreasing the economic impact of locoismon the cattle industry (Ralphs et al., 1984, 1993; James et al.,2005). Swainsonine concentration is measured using liquidchromatography-mass spectrometry (LC-MS) and/or gas

J. Achata Böttger :R. Creamer (*)Department of Entomology, Plant Pathology and Weed Science,New Mexico State University,Las Cruces, NM 88003, USAe-mail: creamer@nmsu.edu

D. GardnerPoisonous Plant Research Lab, USDA ARS,1150 E 1400 N,Logan, UT 84341, USA

J Chem Ecol (2012) 38:486–495DOI 10.1007/s10886-012-0120-z

chromatography–mass spectrometry (GC-MS) (Gardner et al.,2001). Undifilum can be detected by isolation or PCR (Ralphset al., 2008), but these techniques do not produce quantitativeinformation on how much endophyte biomass is present inlocoweed tissues. Quantitative PCR could be used to betterexplain the processes that link endophyte colonization withseasonal fluctuations in the swainsonine content of locoweeds.

Quantitative PCR or real-time PCR (qPCR) has been usefulto describe the extent of colonization of toxigenic fungi in planttissues. Targeting single or low copy number genes throughqPCR makes it possible to quantify Alternaria DNA in planttissue, having a lower detection limit of about one picogram ofpathogen DNA or one genome copy per reaction (Gachon andSaindrenan, 2004; Andersen et al. 2006). By targeting twodifferent single copy genes in the fescue endophyte Neotypho-dium, the lower detection limit of qPCR assays was establishedat 20 genome copies per reaction (Rasmussen et al., 2007); thisquantitative data was used to study the relationship between theextent of endophyte colonization and the synthesis of differentalkaloids in the plant-endophyte system.

Here, we present the development of qPCR assays to assessthe colonization level ofUndifilum in locoweeds.We comparethe extent of endophyte colonization to the swainsonine con-tent in plants grown in vitro, as well as plants collected fromwild populations in New Mexico and Colorado.

Methods and Materials

In Vitro Grown Plants. Oxytropis sericea plants infected withU. oxytropis were cultured in vitro as described in Oldrupet al. (2010), on baseline, acidified and PEG ULT-0 media.Groups of 6 plants were grown in 25 mm deep Petri dishes.Eighteen plants coming from three dishes were pooled andconsidered an individual sample. Five replicates were used permedia type per week during 4 wk. Three independent trials ofthe experiment were performed. Plants were incubated in agrowth chamber at 28°C and 550 μmoles m-2 s-1 continuous

light. Samples were ground to a fine powder with a mortar andpestle using liquid nitrogen. A subsample was immediatelyprocessed for DNA extraction using the DNeasy Plant MiniKit (Qiagen, Inc., Germany), and the rest was desiccated at50°C for 48 h in a drying oven. The dried material was grounda second time with a mortar and pestle to homogenize theparticle size prior to swainsonine extraction.

Field Plant Collections A minimum of 7 healthy-lookinglocoweed plants were sampled from wild populations inNew Mexico and Colorado (Table 1). Plants were flaggedand coded for non-destructive sampling throughout eight sea-sons, from the summer of 2006 to the summer of 2008. Eachsample consisted of 5 leaves, 5 inflorescences, or 5 infrutes-cences collected at the midpoint of each season. Stem sampleswere gathered only when plants were senescent and were notexpected to be found alive the next season. Taxonomic iden-tification was done according to Fox et al. (1998) and underthe guidance of Dr Kelly Allred (Department of Animal andRange Sciences, NMSU).

DNAwas extracted from a composite mixture of leaflet andpetiole fragments, pooled from 5 leaves per each plant. Thesame procedure was used for inflorescences and infrutescences.The rest of each sample was desiccated as mentioned before.Dry samples were ground with a Wiley® mini-millgrinder,sieved through a 0.45 mm mesh screen, and processed forswainsonine extraction.

Real-Time PCR Assays The DNeasy Plant Mini Kit wasused to extract DNA from Undifilum oxytropis isolatedfrom A. mollissimus and O. sericea. Primers ITS5(GCAAGTAAAAGTCGTAACAAGG) (White et al.1990), OR1 (GTCAAAAGTTGAAAATGTGGCTTG),EqR4 (CTGACGCTGATTGCAATTACA), SARF4(GAGAACTCCAGGAGAACTTG), and SARR3(GTGGCAAGATCCTATCCTTC) were used in qPCRassays as follows. Primer set ITS5-OR1 targets the ITS regionand produces a 580 bp fragment and was used for field plants

Table 1 Locoweed populations sampled

Field site - County, State (Habitat) Coordinates and Elevation Sampled area (m2) Species

Jornada del Muerto - Doña Ana Co.,NM (Chihuahuan Desert)

32 °31'46.02"N 106 °46'04.94"W 1333 m 2400 Astragalus mollissimusvar. bigelovii

City of Rocks - Grant Co., NM(Chihuahuan Desert/Grassland)

32 °34'42.05"N 107 °58'21.35"W 1581 m 4000 Astragalus mollissimusvar. bigelovii

Capulin - Union Co., NM(Short grass prairie)

36 °38'14.55"N 103 °56'25.63"W 2062 m 290 Astragalus mollissimus var.mollissimus

Black Range - Grant Co., NM(Montane Conifer Forest)

32 °53'03.97"N 107 °51'48.34"W 2075 m 300 Oxytropis lambertiivar. bigelovii

Capulin - Union Co., NM (Short grass prairie) 36 °38'14.55"N 103 °56'25.63"W 2062 m 290 Oxytropis sericea

Virginia Dale - Larimer Co., CO(Short grass prairie)

40 °54'16.82"N 105 °18'23.15"W 2042 m 400 Oxytropis sericea

J Chem Ecol (2012) 38:486–495 487

only. ITS5 is a universal forward primer and OR1 is a reverseprimer that amplifies both Undifilum and Embellisia. Sincethe ITS5/OR1 amplicon is larger than is commonly used forqPCR, we designed the reverse primer EqR4 to be paired withITS5. This primer set produces a 234 bp amplicon fromUndifilum DNA and can also amplify templates from otherascomycetes. Since ITS5/EqR4 was used for in vitro plants

only, specificity was not a concern. The primer set SARF4/SARR3 was designed to target the U. oxytropis saccharopinereductase gene (sr) (GeneBank HQ010362; Mukherjee andCreamer, unpublished data), producing a 96 bp ampliconunder the same qPCR conditions as ITS5/EqR4.

Twenty microliter reactions were prepared using the DyNA-mo SYBR® Green qPCR kit (Finnzymes, Finland), containing

Table 2 Weather stations in New Mexico and Colorado

Field site Weather station Coordinates Dataa Distance (km) Source

Jornada J. Exp. Range 32 °37'00.00"N 106 °43'60.00"W Temp/Ppt 10.43 NE NOAA

City of Rocks Faywood 32 °37'60.00"N 107 °52'00.00"W Temp/Ppt 12.28 NE NOAA

Capulin Capulin 36 °43'60.00"N 103 °59'60.00"W Temp/Ppt 12.23 NW NOAA

Black Range McKnight cabin 33 °00'00.00"N 107 °52'01.20"W Temp 13.42 N NMCC

Mimbres Ranger STN 32 °55'60.00"N 108 °01'00.00"W Ppt 15.41 NW NOAA

Virginia Dale Virginia Dale 7 ENE 40 °58'00.00"N 105 °13'00.00"W Temp/Ppt 10.13 NE NOAA

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Fig. 1 Real-time PCRquantification of UndifilumDNA standards using primersets ITS5/OR1 (A,D),ITS5/EqR4 (B,E) andSARF4/SARR3 (C,F): Meltingcurves (a, b and c) producedby the two highest workingconcentrations (in ng/μl), thelowest one at whichquantification was attainableand from a control with notemplate. Quantification range(d, e, and d) and regressioncurves (solid line) used tocalculate Undifilum DNAconcentration. Confidenceintervals (dashed lines) forquantification wereestimated with α00.05

488 J Chem Ecol (2012) 38:486–495

2.5 mM MgCl2, 0.625 μM primers, and 1 μl of DNA extract.Assays were carried out in an iQ5 thermal cycler (BioRad,USA). The temperature profile for the reactions consisted of10min at 95°C for initial denaturation, followed by 45 cycles of94°C/10 sec, 54.5°C/20 sec, 72°C/25 sec, and fluorescenceacquisition. This was followed by an extension step at 72°C/10 min, and melting curve acquisition from 65°C to 95°C,reading fluorescence every 0.5° with a hold time of 0.5 sec. Afinal extension step at 72°C/10 min completed the pro-gram. The fluorescence threshold was automatically ad-justed by the iQ5 Optical System Software 2.0 for every

plate. DNA extracted from Undifilum cultures was usedto prepare concentration standards; a four-fold dilutionseries from 1 ng/μl to 61 fg/μl for primer set ITS5/OR1and a ten-fold dilution series from 1 ng/μl to 100 ag/μlfor primer sets ITS5/EqR4 and SARF1/SARR3. Therelationship between the Undifilum DNA concentrationin the standards and their C(t) was used to build a linearequation relating both variables. The C(t) from locoweedDNA samples was used in the equation to solve forUndifilum DNA concentration in each sample. Since the totalDNA concentration is variable between extracts, the Undifi-

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Fig. 3 Undifilum DNA (UD)and swainsonine concentrationin in vitro grown Oxytropissericea plants. Type of mediais indicated by different shapedsymbols as described in thekey. Correlation statisticsare given in the inset

J Chem Ecol (2012) 38:486–495 489

lum DNA concentration was divided by the total DNA con-centration for each individual extract resulting in a ratio we

call “UD” for simplicity. We use the UD ratio as an estimationof the extent of Undifilum colonization in plant tissue.

Table 3 Phenological composition of locoweed populations

Species (location) Season n Phenological composition (%)

Va Flb Fl/Fr Frc

Astragalus mollissimus var. bigelovii Summer 06 12 100 - - -

(Jornada del Muerto, NM) Fall 06 10 100 - - -

Winter 07 10 90 10 - -

Spring 07 10 50 - 20 30

Summer 07 2 100 - - -

Astragalus mollissimus var. bigelovii Summer 06 14 100 - - -

(City of Rocks, NM) Fall 06 14 100 - - -

Winter 07 13 92 8 - -

Spring 07 9 - 11 67 22

Summer 07 4 100 - - -

Fall 07 1 100 - - -

Astragalus mollissimus var. mollissimus Summer 06 7 43 - 29 29

(Capulin, NM) Fall 06 7 100 - - -

Winter 07 5 100 - - -

Spring 07 5 - 100 - -

Summer 07 7 100 - - -

Fall 07 1 100 - - -

Winter 08 2 100 - - -

Oxytropis lambertii var. bigelovii Summer 06 8 25 75 - -

(Black Range, NM) Fall 06 8 25 - - 75

Winter 07 8 100 - - -

Spring 07 8 100 - - -

Summer 07 8 - 75 25 -

Fall 07 7 100 - - -

Winter 08 6 100 - - -

Spring 08 6 100 - - -

Oxytropis sericea Summer 06 7 100 - - -

(Capulin, NM) Fall 06 7 100 - - -

Winter 07 7 100 - - -

Spring 07 7 - 100 - -

Summer 07 7 100 - - -

Fall 07 7 100 - - -

Winter 08 5 100 - - -

Spring 08 2 50 50 - -

Oxytropis sericea Summer 06 4 100 - - -

(Virginia Dale, CO) Fall 06 8 100 0 - -

Spring 07 8 100 - - -

Summer 07 8 100 - - -

Fall 07 7 100 - - -

Winter 08 6 100 - - -

Spring 08 7 71 29 - -

a Vegetative (V), b Flowering (Fl), c Fruiting (Fr)

490 J Chem Ecol (2012) 38:486–495

Swainsonine Extraction and Quantification Swainsoninewas extracted by cationic exchange chromatography(CEC) and quantified by LC-MS (Gardner et al., 2001).Two hundred milligrams of ground plant material were usedfor the extractions. Two hundred microliters of swainsonineextract were evaporated in HPLC vials and rehydrated priorto LC-MS analysis. Swainsonine concentration in the extractswas transformed to “μg of swainsonine per gram of dry plantmatter”.

Weather Data Precipitation and temperature at the field loca-tions were compared to the swainsonine concentration and theUD. Data were downloaded from public records at the NationalOceanic and Atmospheric Administration (NOAA, 2006a, b,2007a, b, 2008a, b, c, d), SNOTEL-Natural Resources Conser-vation Service (NRCS) and the New Mexico Climate Center(NMCC) (Table 2).

Statistical Analysis Undifilum DNA concentration (ng/μl)and swainsonine concentration (μg/ml) were both calculatedin the qPCR and LC-MS instruments using line equationsderived from linear regression. These equations were calcu-lated automatically by the software of each instrument basedon the readings of serial dilutions of a known standard con-centration. The experiment was set up following a three-by-four factorial design. Results were analyzed by two-wayANOVA, using the GLM procedure in SAS 11. Data fromthe in vitro study were subjected to Non-Parametric Pearsoncorrelation analysis to analyze the relationship between Undi-filum DNA and swainsonine contained in the samples.

Significant differences were established atα00.05. All swain-sonine and endophyte DNAmeasurements from field samplesare presented as mean±standard error of the mean (SEM).

Results

Undifilum DNA Quantification Endophyte DNA quantifica-tion using the primer set ITS5/OR1 produced a melting curvewith a main peak at 85.5°C and a secondary peak at 81.5°C(Fig. 1A). The secondary peak could be caused by the lengthof the PCR product and the presence of a high G/C rich regionwithin it. The regression curve calculated from the standardsfollowed a linear trend (r>0.99 in most runs) across theconcentration range used to construct it (Fig. 1D). The lowerquantification limit for this primer set was 61 fg/μl. Amplifi-cation of lower template concentrations can be achieved, but itwas not consistent enough for quantification. Replacing OR1with EqR4 improved amplification sensitivity about a hun-dred fold, detecting Undifilum DNA down to 100 ag/μl(Fig. 1E). However, the linearity of the relationship betweentemplate concentration and C(t) is not maintained over thelower concentrations. Therefore, we established a quantifica-tion limit of 1 fg/μl with the latter primer set. The meltingcurve produced by this amplicon has a single peak at 85.5°C(Fig. 1B). The sr amplicon produced a melting curve with asingle peak at 85°C (Fig. 1C) but the fluorescence signal waslost around 10 pg/μl (Fig. 1F), leaving too narrow a rangefor quantification.

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Fig. 4 Undifilum colonizationin locoweeds and precipitationat the sampling sites: Oxytropissericea in Capulin, NM (a),O. lambertii var. bigelovii (b),Astragalus mollissimus var.mollissimus (c) and A.mollissimus var. bigelovii inCity of Rocks (d). Oxytropissericea in Virginia Dale and A.mollissimus var. bigelovii inJornada del Muerto weresimilar to their counterpartsand thus their graphs wereomitted. Samplingseasons are abbreviated byfirst two letters and year

J Chem Ecol (2012) 38:486–495 491

In vitro O. sericea. The Undifilum colonization increasedover time, as indicated by the UD, reaching levels around 1 pgUndifilum DNA/ng of total DNA on all three media at the endof the fourth week (Fig. 2A). Plants grown in acidified andPEG ULT-0 showed colonization levels as high on the secondweek as plants grown in regular ULT-0 did on the fourth week.In general, UD was quite variable during the first three weeks,but it became more stable by the fourth week. Swainsonineconcentration also increased over time, and it was even moreinfluenced by the different media than endophyte colonization(Fig. 2B). Interestingly, the acidification of the media resultedin extremely low swainsonine content during the four weeks.Correlation between Undifilum DNA and swainsonine con-tent shows that in general, higher levels of colonization trans-late into higher swainsonine content (Fig. 3). However, thiscorrelation was high and significant only in plants grown onULT-0 (ρ00.972, P<0.001).

Field Plants Locoweeds were found in the vegetative statethroughout most of the sampling seasons (Table 3); however,most individuals did produce seed as indicated by empty podsfound still attached to the plants during summer and fall. Thesample size decreased over time as some plants senesced anddied, which occurred earlier and more extensively with Astra-galus when compared to Oxytropis. The extent of endophytecolonization in leaves ranged from approximately 0.1 to 10 pgUndifilumDNA/ng of total DNA (Fig. 4). Colonization can bestable and be maintained within the same order of magnitude

as inO. lambertii and A.mollissimus var.mollissimus (Fig. 4Band C). It can also change as much as a hundred fold withintwo seasons as in O. sericea and A. mollissimus var. bigelovii(Fig. 4 A and D). The seasonal changes in swainsoninecontent hinted at more of an annual cycle, with ranges moreproperly defined by the specific locoweed-endophyte associ-ation (Fig. 5). Astragalus mollissimus var. bigelovii fromJornada del Muerto exhibited a highly significant strong cor-relation between colonization and swainsonine content (ρ00.5439; P<0.001), where as O. lambertii and O. sericea fromVirginia Dale exhibited significant, however moderate corre-lations (Table 4). The coefficients indicate that colonization isfar from fully explaining the content of swainsonine in a plant.The average daily temperature was found to be stronglycorrelated with the average swainsonine content in leaves of

Table 4 Non-parametric correlation analysis between Undifilum coloni-zation and swainsonine content in field plants

Population n ρ1 P value

Oxytropis lambertii var. bigelovii (BR) 65 0.3008 0.0149

O. sericea (C) 45 0.0586 0.7020

O. sericea (VD) 40 0.3989 0.0108

Astragalus mollissimus var. bigelovii (JM) 41 0.5439 0.0002

A. mollissimus var. bigelovii (CR) 60 0.0923 0.4830

A. mollissimus var. mollissimus (C) 27 0.2076 0.2989

1 Spearman’s correlation coefficient

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Fig. 5 Swainsonine content inlocoweeds and environmentaltemperature at the sampling sites:Oxytropis sericea in Capulin, NM(a),O. lambertii var. bigelovii (b),Astragalus mollissimus var.mollissimus (c) and A.mollissimus var. bigelovii in Cityof Rocks (d).Oxytropis sericea inVirginia Dale and A. mollissimusvar. bigelovii in Jornada delMuerto were similar to theirvarietal counterparts and thustheir graphs omitted. Averagedaily temperature during thesampling month (dashed line) isshown as well as the highest andlowest temperatures recorded forthe same month (solid line anddotted line respectively).Sampling seasons are abbreviatedby first two letters and year

492 J Chem Ecol (2012) 38:486–495

O. lambertii (ρ0−0.8743; P00.004) and A. mollissimus var.mollissimus (ρ00.9; P00.038), outlining seasonal swainso-nine content cycles for these two populations (Table 5 andFig. 6).

Discussion

Using sr to quantify Undifilum DNA results in a qPCR assaysensitive down to picograms per microliter, which is similar towhat is used to detect Alternaria or Neotyphodium using lowor single-copy genes (Gachon and Saindrenan, 2004; Ander-sen et al. 2006; Rasmussen et al., 2007). In the case ofAlternaria, such a detection limit is roughly equivalent toeighty genome copies per reaction (Andersen et al. 2006).Unlike sr, the ITS region occurs in hundreds of copies pergenome in fungi (Kobayashi, 2011), which allowed us todesign qPCR assays orders of magnitude more sensitive,and just as accurate. The two assays we developed allowaccurate quantification down to the order of femtograms ofUndifilum DNA per reaction. Considering the data on Alter-naria (Andersen et al. 2006), we speculate that this quantifi-cation limit could be equivalent to just a fraction of a genome

per reaction. If correct, that would explain the successful butinconsistent amplification of endophyte DNA between 100attograms and 10 femtograms per reaction.

This is not the first time the ITS region has been used toinfer fungal biomass in plant tissues. Glynn et al. (2007)designed competitive PCR assays to quantify Fusarium andMicrodochium infection in wheat seeds. The assay theydesigned is just as sensitive as that presented here; however,it requires two primer sets per reaction as well as visualiza-tion of the PCR products to interpret the outcome. Recently,Cook et al. (2009) reported a qPCR assay to quantify Undi-filum DNA in locoweeds, also based on ITS amplification.They pointed out the need for an assay more sensitive than10 pg Undifilum DNA per microliter, which we provide hereby three orders of magnitude.

The concentration of endophyte DNA in the plant tissuesappears to be a good indicator of the extent of the endophytecolonization. In the in vitro assays, colonization in O. sericeaincreased in the first four weeks after germination, independentof the stress that was simulated. Similarly, swainsonine in-creased over time, and it was detected at levels above baselinebetween the second and fourth week of development. In plantsgrown on baseline medium, the synthesis of swainsonine canbe directly attributed to increasing fungal biomass. However,when plants were grown inmodified media, the correlation waslost, and swainsonine production seemed to be delayed. Be-sides this lag, the relationship between colonization and theswainsonine content in the plant tissue was overall positive. Atweek four, the swainsonine concentration reached the maxi-mum of 67.71±15.51μg/g dry weight, similar to the 73 μg/greported in eight-week-old plants grown under similar condi-tions (Oldrup et al., 2010).

The swainsonine content range in the populations sam-pled is similar to previous reports (Braun et al., 2003;Valloton and Sterling, 2007; Parker, 2008; Ralphs et al.,2008). The populations that showed the highest swainsoninecontent were from varieties known to be highly toxic. In

Olb-BR

Os-C

Os-VD

Amb-JM

Amb-CR

Amm-C

0

500

1000

-5 -4 -3 -2 -1 0

Sw

ains

onin

e (μ

g/g)

Log10 (UD)

Fig. 6 Undifilum DNA and swainsonine contents from six locoweedpopulations (Mean±SEM). Abbreviations correspond to locoweed speciesand location. Astragalus mollissimus var. bigelovii (Amb), Astragalus

mollissimus var mollissimus (Amm), Oxytropis lambertii var. bigelovii(Olb), Oxytropis sericea (Os); Black Range (BR), Capulin (C), VirginiaDale (VD), City of Rocks (CR), Jornada del Muerto (JM)

Table 5 Non-parametric correlation between the average dailytemperature at the sampling sites and the average swainsonine contentin leaves

Population n ρ1 P value

Oxytropis lambertii var. bigelovii (BR) 8 −0.8743 0.0045

O. sericea (C) 7 0.6071 0.1482

O. sericea (VD) 7 0.3214 0.4821

Astragalus mollissimus var. bigelovii (JM) 5 −0.7000 0.1881

A. mollissimus var. bigelovii (CR) 6 −0.5429 0.2657

A. mollissimus var. mollissimus (C) 5 0.9000 0.0378

1 Spearman’s correlation coefficient

J Chem Ecol (2012) 38:486–495 493

addition, during this sampling period, they were subjected tothe most extreme environmental temperature differentialswithin the group. Water deficiency has been considered alikely elicitor of the swainsonine production pathway, butalthough rainfall was low in both years, the swainsoninecontent in A. mollissimus var. mollissimus was below theconcentration Valloton and Sterling (2007) associate withdrought stress.

Here, an annual cycle for toxicity in the field is proposedbased on the data on leaf tissue of A. mollissimus var. mollis-simus and O. sericea from Capulin, NM. Overwinteringleaves have the lowest swainsonine concentrations; swainso-nine content then increases during the spring as snow meltsand the plants awaken from dormancy. Plants develop newfoliage and start blooming early in the spring, then developfruits that ripen before mid-summer. Meanwhile, swainsoninecontent increases in the leaves, without a major change in theextent of endophyte colonization, reaching amaximum swain-sonine concentration around mid-summer, after seed dispers-al. Frosts and cold wind sweep the pasture and leaves startsenescing during the first half of fall, but they stay attached tothe plant. Dead dry leaves and petioles protect the few leavesproduced in the middle of the plant at the end of summer. Theswainsonine content in the last leaves of summer is lowercompared to the fully developed leaves around them, but theendophyte colonization is not different. During winter snowcovers, the ground and the plants stay dormant, with fewprimordial leaves alive. Those overwintering leaves have thelowest swainsonine content throughout the year, but they arethe same leaves that were produced at the end of summer,surviving through the fall. Since swainsonine was there at thebeginning, it is potentially being degraded, transformed, ortranslocated to the underground tissues. The cycle is restartedas winter progresses onto spring and plants awaken fromdormancy, and they either produce more swainsonine, translo-cate it from underground tissues or both.

It was not a surprise to find that locoweed toxicity changedover time, in part because toxicity data exist for those varietiesand because research has been done over the last two decadeson some of the populations we analyzed. Not only is the extentof endophyte colonization variable, but it also appears to beresponsive to environmental cues. Using the amount of Undi-filum DNA as a fair representation of the endophytic biomass,we are reporting a range of ten to a hundred-fold the amount ofthe symbiont biomass inside hosts belonging to the samepopulation.

Swainsonine synthesis is influenced by the extent of endo-phyte colonization in the weeks following germination. Theextent of plant colonization found at the end of the fourthweek is similar to that found in mature plants from the field.This suggests that geographic location and/or weather condi-tions influence swainsonine production by locoweeds veryearly in plant development. Additional work will be necessary

to elucidate the role of weather parameters such as tempera-ture extremes on swainsonine production.

Acknowledgements We thank Dr Jose Valdez-Barillas and DavidGraham for their guidance during field seasons, and Dr Steve Hanson,Dr Champa Sengupta-Gopalan, and Dr Tracy Sterling for allowing theuse of their lab facilities and equipment. We acknowledge USDASpecial Grant # 59-5428-1-327 and the New Mexico State UniversityAgricultural Experiment Station for supporting this work.

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